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Knowledge and Management of Aquatic Ecosystems (2014) 414, 12 http://www.kmae-journal.org c ONEMA, 2014 DOI: 10.1051/kmae/2014024 Infestation of Lernaea cyprinacea (Copepoda: Lernaeidae) in two invasive fish species in Romania, Lepomis gibbosus and Pseudorasbora parva M.M. Stavrescu-Bedivan(1),O.P.Popa(2),L.O.Popa(2), Received May 14, 2014 Revised July 14, 2014 Accepted July 17, 2014 ABSTRACT Key-words: In this study we analyzed comparatively the host-parasite associations non-indigenous between two fish host species invasive in Europe (Lepomis gibbosus and fish, Pseudorasbora parva) and one known generalist parasite species, the host-parasite copepod Lernaea cyprinacea. We used a fragment of the hypervariable coevolution, region D1-D2 of the 28S rRNA to confirm that the copepod specimens quantitative collected on both host species in our study are indeed conspecific. The parasitology, prevalence of infection was significantly different between the two host DNA barcoding, species in all three aquatic ecosystems. Two populations of L. gibbosus molecular exhibited a positive correlation coefficient between the standard body markers length and infection intensity, while a negative correlation coefficient was observed in one population of P. par v a. This is one of the few studies pro- viding parasitological parameters of infections of Lernaea cypriancea in Lepomis gibbosus and Pseudorasbora parva. RÉSUMÉ Infestation de Lernaea cyprinacea (Copépode : Lernaeidae) sur des espèces envahis- santes de poissons Lepomis gibbosus et Pseudorasbora parva en Roumanie Mots-clés : Dans cette étude, nous avons analysé comparativement les associations hôte- poissons parasite entre deux espèces hôtes de poissons envahissantes en Europe (Lepomis non indigènes, gibbosus et Pseudorasbora parva) et une espèce de parasite généraliste connue, coévolution le copépode Lernaea cyprinacea. Nous avons utilisé un fragment de la région hy- hôte-parasite, pervariable D1-D2 de l’ARNr 28S pour confirmer que les échantillons de copé- parasitologie podes prélevés sur les deux espèces hôtes dans notre étude sont en effet de la quantitative, même espèce. La prévalence de l’infection était significativement différente entre ADN barcoding, les deux espèces hôtes dans les trois écosystèmes aquatiques. Deux populations de L. gibbosus présentaient un coefficient de corrélation positif entre la longueur marqueurs standard du corps et l’intensité de l’infection, tandis qu’un coefficient de corréla- moléculaires tion négatif a été observé dans une population de P. p a r v a . C’est l’une des rares études fournissant des paramètres parasitologiques de l’infection de Lernaea cy- priancea sur Lepomis gibbosus et Pseudorasbora parva. (1) Faculty of Agriculture, University of Agronomic Sciences and Veterinary Medicine of Bucharest, Bucharest, Romania (2) Molecular Biology Department, “Grigore Antipa” National Museum of Natural History, 011341 Bucharest, Romania Corresponding author: [email protected] Article published by EDP Sciences M.M. Stavrescu-Bedivan et al.: Knowl. Managt. Aquatic Ecosyst. (2014) 414, 12 INTRODUCTION Host-parasite coevolution may lead to either host or parasite local adaptation in spatiotem- porally varying environments, e.g. when invasive species expand their range (Lemoine et al., 2012). One of the factors considered to explain the success of these species is the release of the newly founded populations from the pressure exercised by their original parasites left be- hind in the initial environment (Mastitsky et al., 2014;Torchinet al., 2003). This theory relies on the idea that co-evolution leads to extensive local adaptations, and as such the parasites in a particular area infect hosts from the same area more efficiently than they infect hosts from an- other geographically distinct population (Morand et al., 1996;Rothet al., 2012). On the other hand, since the newly conquered environments are not parasite-free, the new host-parasite interactions can lead to parasite maladaptation, where parasites can temporarily have higher fitness in the newly arrived (allopatric) hosts than in the sympatric hosts (Dybdahl and Storfer, 2003). In this study we analyzed comparatively the host-parasite associations between two fish host species invasive in Europe (Lepomis gibbosus and Pseudorasbora parva) and one known generalist parasite species, the copepod Lernaea cyprinacea. Lepomis gibbosus (Linnaeus, 1758) is a freshwater fish species native to North America, be- longing to the family Centrarchidae. Introductions of pumpkinseed sunfish beyond its native range have occurred around the world for many years, and for many reasons, such as poten- tial sport, ornamental pond fish, fry and fingerlings, and prey for native wild stocks (Jordan, 2009). Introduced to Europe in the 1880s as a pond and aquarian fish, the species is quite widespread throughout this continent, being particularly abundant in Mediterranean coun- tries (Hanel et al., 2011). The second host in our study, Pseudorasbora parva (Temminck & Schlegel, 1846), is an Asian native freshwater cyprinid species considered a typical invasive species and occurring on all continents except for Antarctica (Margaritov and Kiritsis, 2011). The accidental transfer and release of P. par v a with carp species translocations for aquacul- ture is considered the primary pathway of species introduction into new areas (Gozlan, 2012). After the first introduction into Europe in the 1960s (Nucet, Romania), the species extended its range through natural dispersal, recreational fishing and ornamental fish trade. Its high pheno- typic plasticity and the ability to cope with novel pathogens have predisposed the topmouth gudgeon to being a strong invader (Hanel et al., 2011). The parasite species, the anchor worm Lernaea cyprinacea Linnaeus, 1758, is the only cosmopolitan species in the genus Lernaea (Piasecki et al., 2004). This copepod is non-specific, infecting many freshwater fish species (Williams and Bunkley-Williams, 1996), but also affecting marine fish hosts (Gutierrez-Galindo and Lacasa-Millan, 2005), as well as adult and larval amphibians (Kupferberg et al., 2009; Nagasawa et al., 2007) and larval aquatic insects (McAllister et al., 2011). L. cyprinacea is native to Asia, but currently it is widely distributed (Oscoz, 2010). The aim of the present study was to compare the parasitological parameters of L. cyprinacea infections on the two invasive fish species in several lentic systems in Romania. MATERIALS AND METHODS We conducted parasite surveys of fish fauna during the 2008 summer season in three Ro- manian lentic ecosystems: Moara Domneasca Lake, Ilfov County – 46◦4356N, 27◦4441E (2 surveys in June and July); Brates ¸ Lake, Gala¸ti County – 45◦2859N, 28◦413E(1survey in July); Crapina Lake, Tulcea county – 45◦2224N, 28◦1442E (1 survey in July). The fish specimens were collected by electrofishing with a Samus 720 MP device (Samus Special Electronics, Poland) and immediately after the sampling, they were placed in 95% ethanol and transported to the laboratory. Further analyses of parasite fauna were performed only on L. gibbosus and P. par v a, because both species exhibited a consistent abundance in all samples. For each fish specimen, the standardized length was measured with a calliper to the nearest 0.1 mm. Each fish specimen was examined using a Krüss Optronic dissecting microscope at the level of the branchial cavities, head, fins and skin for presence of copepod 12p2 M.M. Stavrescu-Bedivan et al.: Knowl. Managt. Aquatic Ecosyst. (2014) 414, 12 parasites. The copepod specimens removed were placed in 95% ethanol for further analysis. The morphological identification of the L. cyprinacea specimens was performed according to Bauer (1987). The copepod preference for a particular attachment site on the host was recorded and 95% confidence intervals of the parasite proportions on different host regions were computed with the modified Wald method (Agresti and Coull, 1998). The infestation parameters prevalence, mean intensity, median intensity and mean abundance, as well as the corresponding 95% C.I., were calculated using the software package Quantitative Para- sitology 3.0 (Rozsa et al., 2000). The Spearman rank correlation coefficient (rs) was used to measure correlation between host fish size and the abundance of infection, while a p-value for the null hypothesis of rs being equal to zero was computed based on 10000 Monte Carlo replications using the same software package. The comparison of prevalence between popu- lations of the same host species and between host species in the same lake were performed by Fisher’s exact test. In all cases p values lower than 0.05 were considered significant. We tested that the copepod individuals collected on both fish hosts are conspecific (L. cypri- nacea) by DNA sequencing of a fragment of the 28S rDNA. Total DNA was extracted from eight copepod individuals, from both host species, using the NucleoSpinR Tissue kit (Macherey–Nagel GmbH & Co. KG, Düren, Germany), according to the manufacturer’s specifications. The amplification of a fragment of the hypervariable region D1-D2 of the 28S rDNA fragment was performed with the primers 28SF (5–acaactgtgatgcccttag–3) and 28SR (5–tggtccgtgtttcaagacg–3)(Songet al., 2008). The amplification was performed in a 50 µL reaction containing 10 mM Tris–HCl (pH 8.8 at 25 ◦C), 50 mM KCl, 0.08% (v/v) Nonidet P40, 2.5 mM MgCl2, each dNTP at 0.1 mM, each primer at 0.2 µM, 1 unit of Taq DNA polymerase (Fermentas UAB, Vilnius, Lithuania), and approximately 50 ng of DNA template. The temper- ature profile of the polymerase chain reaction for the 28S marker consisted of initial denatu- ration at 94 ◦C for 30s, followed by 30 cycles at 93 ◦C for 30s, 50 ◦C for 45s, 72 ◦C for 45s, and a final extension step performed at 72 ◦C for 10 min. The PCR products were separated on 1% agarose gel, and the fragments of interest purified using the innuPREP DOUBLEpure Kit (Analytik Jena AG, Jena, Germany). The DNA sequencing was performed by Macrogen Europe (1105 AZ, Amsterdam). RESULTS During the 2008 summer surveys 312 specimens of Lepomis gibbosus and 393 specimens of Pseudorasbora parva were collected.

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